Human Amyloidogenic Light Chains Directly Impair Cardiomyocyte Function Through an Increase in Cellular Oxidant Stress
Primary amyloidosis is a systemic disorder characterized by the clonal production and tissue deposition of immunoglobulin light chain (LC) proteins. Congestive heart failure remains the greatest cause of death in primary amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Amyloid cardiomyopathy is largely unresponsive to current heart failure therapies, and is associated with a median survival of less than 6 months and a 5-year survival of less than 10%. The mechanisms underlying this disorder, however, remain unknown. In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy (cardiac-LC), specifically alter cellular redox state in isolated cardiomyocytes, marked by an increase in intracellular reactive oxygen species and upregulation of the redox-sensitive protein, heme oxygenase-1. In contrast, vehicle or control LC proteins isolated from patients without cardiac involvement did not alter cardiomyocyte redox status. Oxidant stress imposed by cardiac-LC proteins further resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intracellular calcium handling. Cardiomyocyte dysfunction induced by cardiac-LC proteins was independent of neurohormonal stimulants, vascular factors, or extracellular fibril deposition, and was prevented through treatment with a superoxide dismutase/catalase mimetic. This study suggests that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.
Primary (AL) amyloidosis is a plasma cell dyscrasia resulting in the clonal production of immunoglobulin light chain proteins (LC) and subsequent multi-organ dysfunction.1 Congestive heart failure remains the greatest cause of death in AL amyloidosis, due to the development of a rapidly progressive amyloid cardiomyopathy. Patients with amyloid cardiomyopathy are largely unresponsive to current heart failure therapies2,3 and have a median survival of less than 6 months and a 5-year survival of less than 10%.3,4 The mechanisms underlying this disorder, however, have yet to be determined. Prior theories have suggested that interstitial fibril deposition of AL proteins are the main cause of contractile dysfunction and cardiomyopathy.2 This, however, is inconsistent with clinical observations, which have detailed a lack of correlation between myocardial fibril deposition and cardiac dysfunction in AL patients.5 In addition, recent work in noncardiac tissues have suggested that other amyloidogenic proteins, including Aβ and transthyretin, may directly impair cellular function, independent of fibril deposition, through redox sensitive mechanisms.6,7
In this report, we demonstrate that physiological levels of human amyloid LC proteins, isolated from patients with amyloid cardiomyopathy, specifically alter cellular redox state in cardiomyocytes, marked by an increase in intracellular reactive oxygen species (ROS) and upregulation of the redox-sensitive protein, heme oxygenase-1 (HO-1). Oxidant stress imposed by cardiac-LC proteins resulted in direct impairment of cardiomyocyte contractility and relaxation, associated with alterations in intracellular calcium handling. Cardiomyocyte dysfunction was also found to be independent of neurohormonal stimulants, vascular factors, and extracellular fibril deposition, and was prevented through antioxidant treatment.
Materials and Methods
Cultured adult rat ventricular cardiomyocytes were treated with vehicle, control LC isolated from patients with nonamyloidogenic myeloma and noncardiac-involved AL amyloidosis (Con-LC), or LC isolated from patients with cardiac-involved AL amyloidosis (Cardiac-LC) at physiological concentrations (20 mg/L) for 24 hours. Antioxidant experiments were conducted with the superoxide dismutase/catalase mimetic, MnTMPyP. Cellular redox status was determined using dichlorofluorescein-diacetate fluorescence and HO-1 protein expression. LC aggregation and amyloid fibril formation were also assessed.8 Cardiomyocyte contractility and intracellular calcium transients were determined in cells paced at 5 Hz at 37°C.9
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
Cardiac-LC Increase Intracellular ROS- and Redox-Sensitive Protein Expression
To determine whether physiological levels of LC directly alter redox status in cardiomyocytes, intracellular ROS levels were determined after exposure to amyloidogenic proteins. As shown in Figure 1A, human Con-LC proteins did not influence ROS levels relative to vehicle exposure. In contrast, Cardiac-LC proteins directly induced cellular oxidant stress, as demonstrated by enhanced DCF fluorescence. Elevated ROS levels in Cardiac-LC cells were prevented through antioxidant treatment with MnTMPyP.
Redox-sensitive protein expression was also determined after incubation with LC, using the cellular stress marker, HO-1.10 Cardiac-LC resulted in an upregulation of HO-1 expression relative to vehicle and Con-LC in a dose-dependent manner (Figure 1B). Interestingly, even low levels of Cardiac-LC (10 mg/L) increased HO-1 expression, and this effect was augmented greater than 2-fold with physiological concentrations of LC protein. In addition, HO-1 induction increased in a time-dependent manner with Cardiac-LC (online Figure available in the online data supplement), with augmented expression as early as 6 hours after incubation. Induction of HO-1 was prevented through antioxidant treatment with the superoxide dismutase/catalase mimetic, MnTMPyP, confirming redox-sensitive expression (Figure 1B).
Importantly, LC proteins did not form either LC aggregates or amyloid fibrils, as determined by spectrophotometric analysis8 (Figure 1C) or Congo red staining (data not shown), suggesting that Cardiac-LC directly altered redox state in cardiomyocytes, independent of fibril formation.
Cardiac-LC Impairs Cardiomyocyte Contractility and Intracellular Calcium Handling
We subsequently determined the effects of LC and altered redox state on cardiomyocyte contractility and intracellular calcium transients under physiological conditions. Cardiac-LC directly reduced cardiomyocyte cell shortening (Figure 2A) and prolonged cellular relaxation (Figure 2B), whereas Con-LC did not alter cellular function. As shown in Figure 2C, decreased cell shortening with Cardiac-LC was accompanied by a concomitant decrease in both peak intracellular calcium levels and calcium transient amplitude, thereby suggesting that Cardiac-LC mediated contractile dysfunction was secondary to impaired calcium handling. Interestingly, however, altered cellular relaxation with Cardiac-LC was not associated with prolonged intracellular calcium reuptake (Figure 2D), consistent with a calcium-independent mechanism for impaired relaxation. The effects of Cardiac-LC proteins on cardiomyocyte function and intracellular calcium were reversed to baseline levels with antioxidant treatment, suggesting that Cardiac-LC induced cellular dysfunction was secondary to increased oxidant stress. Representative tracings of cell shortening and intracellular calcium transients are shown in Figure 2E.
Amyloid cardiomyopathy represents a rapidly progressive and fatal form of cardiomyopathy. The mechanisms underlying this disease remain unknown. In this report, we demonstrate that human amyloidogenic cardiac-LC proteins specifically alter cellular redox state in isolated cardiomyocytes, resulting in direct impairment of cardiomyocyte contractile function and calcium handling.
This is the first report to document a direct effect of cardiac-LC on cardiomyocyte redox status and contractile function. Decreased cellular shortening in cardiomyocytes with cardiac-LC treatment was accompanied by a decrease in calcium release during contraction. Impaired cellular relaxation, however, was not associated with slowed calcium reuptake. Therefore, whereas dysfunction of cardiomyocyte contraction and relaxation were both redox-sensitive and reversed with antioxidant treatment, this dysfunction was likely secondary to intracellular calcium-dependent as well as calcium-independent mechanisms. Impaired cellular function may represent oxidant stress-mediated modification of redox-sensitive calcium handling proteins and/or myofilament proteins.11 Our observation of impaired contraction and relaxation in cardiomyocytes is in contrast to prior work detailing an effect of cardiac LC only on diastolic function in isolated hearts.12 This discrepancy, however, is likely secondary to differences in methodology (loaded isovolumically-contracting hearts versus unloaded cardiomyocytes) and cardiac-LC exposure time in isolated hearts versus cardiomyocytes (30 minutes versus 24 hours, respectively).
Importantly, the effects of cardiac-LC occurred in the absence of neurohormonal/vascular factors or extracellular fibril deposition, previously believed to be central to the pathogenesis of amyloid cardiomyopathy. As such, these results may explain clinical observations detailing a discrepancy between the degree of myocardial amyloid fibril deposition and cardiac dysfunction.5 Interestingly, only LC proteins associated with cardiomyopathy, rather than noncardiac-associated LC proteins, resulted in increased ROS and cardiomyocyte dysfunction in isolated cells, suggesting that LC primary sequence and/or posttranslational modifications, rather than osmolar stress or nonspecific protein-receptor interaction, dictate end-organ targeting and dysfunction. Moreover, significant cellular oxidant stress and cardiomyocyte dysfunction were observed with physiological concentrations of LC proteins, and with proteins isolated from multiple patients, thereby eliminating artifact associated with supranormal protein concentrations or single protein variants. These results are consistent with prior findings documenting direct impairment of cellular function, independent of fibril deposition, in noncardiac tissue by other amyloidogenic proteins.6,7
The heightened sensitivity of cardiomyocytes to oxidant injury has been well documented, and the role of ROS in the pathogenesis of myocardial failure continues to expand.13 Furthermore, emerging evidence suggests that increased oxidant stress and oxidative cellular injury contribute significantly to the pathophysiology of other amyloidogenic disease, notably Alzheimer’s disease.7 The mechanisms by which amyloid cardiac-LC proteins increase cellular ROS alter redox state may include activation of cellular oxidase complexes, mitochondrial dysfunction, or metal ion reduction.7,14
This study suggests for the first time that cardiac dysfunction in amyloid cardiomyopathy is directly mediated by LC protein-induced cardiomyocyte oxidant stress and alterations in cellular redox status, independent of fibril deposition. Antioxidant therapies or treatment strategies aimed at eliminating circulating LC proteins may therefore be beneficial in the treatment of this fatal disease.
This study was supported by grants from the Evans Medical Foundation, Gerry Foundation, Young Family Amyloid Research Fund, and NIH grants HL-68705 (M.S.) and HL-67297 and HL-73756 (R.L.). The authors thank Violet Roskens and Jeremy Eberhard for assistance with protein purification.
Original received January 20, 2004; revision received March 15, 2004; accepted March 16, 2004.
Braunwald E. Heart Disease. 5th ed. Philadelphia, Pa: W.B. Saunders Company; 1997.
Dubrey SW, Cha K, Skinner M, LaValley M, Falk RH. Familial and primary (AL) cardiac amyloidosis: echocardiographically similar diseases with distinctly different clinical outcomes. Heart. 1997; 78: 74–82.
Jiang X, Buxbaum JN, Kelly JW. The V122I cardiomyopathy variant of transthyretin increases the velocity of rate-limiting tetramer dissociation, resulting in accelerated amyloidosis. Proc Natl Acad Sci U S A. 2001; 98: 14943–14948.
Yet SF, Tian R, Layne MD, Wang ZY, Maemura K, Solovyeva M, Ith B, Melo LG, Zhang L, Ingwall JS, Dzau VJ, Lee ME, Perrella MA. Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ Res. 2001; 89: 168–173.
Liao R, Jain M, Teller P, Connors LH, Ngoy S, Skinner M, Falk RH, Apstein CS. Infusion of light chains from patients with cardiac amyloidosis causes diastolic dysfunction in isolated mouse hearts. Circulation. 2001; 104: 1594–1597.